Spontaneous Cell Fusion in Macrophage Cultures Expressing High Levels of the P2Z/P2X7 Receptor

Mouse and human macrophages express a plasma membrane receptor for extracellular ATP named P2Z/P2X₇. This molecule, recently cloned, is endowed with the intriguing property of forming an aqueous pore that allows transmembrane fluxes of hydrophylic molecules of molecular weight below 900. The physiological function of this receptor is unknown. In a previous study we reported experiments suggesting that the P2Z/P2X₇ receptor is involved in the formation of macrophage-derived multinucleated giant cells (MGCs; Falzoni, S., M. Munerati, D. Ferrari, S. Spisani, S. Moretti, and F. Di Virgilio. 1995. J. Clin. Invest. 95:1207– 1216). We have selected several clones of mouse J774 macrophages that are characterized by either high or low expression of the P2Z/P2X₇ receptor and named these clones P2Zhyper or P2Zhypo, respectively. P2Zhyper, but not P2Zhypo, cells grown to confluence in culture spontaneously fuse to form MGCs. As previously shown for human macrophages, fusion is inhibited by the P2Z/P2X₇ blocker oxidized ATP. MGCs die shortly after fusion through a dramatic process of cytoplasmic sepimentation followed by fragmentation. These observations support our previous hypothesis that the P2Z/P2X₇ receptor is involved in macrophage fusion.     

Sex-related differences in first-stage impedance cardiographic variables

Impedance cardiography (ZCG), a noninvasive technique used to determine stroke volume and ventricular performance, is currently being utilized more extensively in psychophysiological research and may also prove a useful tool in the control of hypertension. To date, most studies employing ZCG have tested male subjects or patients. However, women and men differ on two important parameters used to calculate stroke volume: thoracic impedance (Zo), and the first derivative of change in impedance over time (dZ/dt_min). Examination of the clinical records of 19 females and 19 males, all nonmedicated, indicated that women had higher Zo levels (F(1, 36)=46.2,p<.0001) and higher dZ/dt_min levels (F(1, 36)=51.8, p<.0001), although calculated stroke volume indexes did not differ. A second sample of 10 women and 8 men, all healthy and nonmedicated, confirmed these findings. The differences in Zo are not due entirely to sex differences in height, weight, percent body fat, and subscapula skin-fold thickness, although these factors differ across sexes. It is important for researchers and clinicians utilizing ZCG with female subjects to be aware of these differences.     

Sources of variation in response intervals after prostaglandin treatment in mares with functional corpora lutea.

Sixty-two non-cycling mares were classified according to the size of largest follicles at the time of treatment with Prostalene, an analogue of prostaglandin (PG) F-2 alpha. Although oestrus occurred in only 77.4% of mares, 98.4% ovulated at an average of 6.8 days after treatment. Greatest variance of interval to ovulation was observed in mares having follicles greater than or equal to 40 mm at the time of treatment. This was due to regression of large follicles about one-third of the time and later ovulation of a succeeding follicle. This resulted also in greatest uncertainty of prediction of ovulation time based on ovarian palpation. Ninety foaling mares were given Prostalene at various days following the first ovulation post partum. Ovulation less than 6 days after treatment was strongly associated with the presence of a large follicle on the day of treatment. Otherwise most (72%) ovulations occurred 6--10 days after treatment. The distribution of interovulatory periods resulting from Prostalene on Day 6 after ovulation differed from that of Day 8 treatment.     

Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.     

Comparative Genomics Reveals Insight into Virulence Strategies of Plant Pathogenic Oomycetes

The kingdom Stramenopile includes diatoms, brown algae, and oomycetes. Plant pathogenic oomycetes, including Phytophthora, Pythium and downy mildew species, cause devastating diseases on a wide range of host species and have a significant impact on agriculture. Here, we report comparative analyses on the genomes of thirteen straminipilous species, including eleven plant pathogenic oomycetes, to explore common features linked to their pathogenic lifestyle. We report the sequencing, assembly, and annotation of six Pythium genomes and comparison with other stramenopiles including photosynthetic diatoms, and other plant pathogenic oomycetes such as Phytophthora species, Hyaloperonospora arabidopsidis, and Pythium ultimum var. ultimum. Novel features of the oomycete genomes include an expansion of genes encoding secreted effectors and plant cell wall degrading enzymes in Phytophthora species and an over-representation of genes involved in proteolytic degradation and signal transduction in Pythium species. A complete lack of classical RxLR effectors was observed in the seven surveyed Pythium genomes along with an overall reduction of pathogenesis-related gene families in H. arabidopsidis. Comparative analyses revealed fewer genes encoding enzymes involved in carbohydrate metabolism in Pythium species and H. arabidopsidis as compared to Phytophthora species, suggesting variation in virulence mechanisms within plant pathogenic oomycete species. Shared features between the oomycetes and diatoms revealed common mechanisms of intracellular signaling and transportation. Our analyses demonstrate the value of comparative genome analyses for exploring the evolution of pathogenesis and survival mechanisms in the oomycetes. The comparative analyses of seven Pythium species with the closely related oomycetes, Phytophthora species and H. arabidopsidis, and distantly related diatoms provide insight into genes that underlie virulence.     

Plasma constituents and mortality in rat pups given chronic insulin via injection, pellet, or osmotic minipump

Two studies compared the glucose responses of 9-day-old rats given subcutaneous insulin, either continuously or via daily injection, for 10 days. In Experiment 1, implanted pellets released a total of 0, 1.9, or 5.7 U insulin/kg the first 24 h. Injected doses were larger, 0 or 8 U/kg. Injections caused no deaths, but insulin-releasing pellets caused high mortality within 24 h. Pups surviving the pellets were normoglycemic by treatment day 8. In Experiment 2, pups received 0.184 U of insulin daily, approximately 8 U/kg at 9 days, via either injection or osmotic minipump. All pups survived. Injected pups were hypoglycemic 2 h postinjection through treatment day 10, whereas pups with insulin minipumps were normoglycemic by day 5. Insulin injections, but not minipumps, lowered plasma triglycerides on day 10. To examine age differences in response to insulin, additional pups and adults received daily injections of 0 or 8 U/kg for 10 days. All survived. Insulin lowered plasma glucose more in pups than in adults and reduced triglycerides in pups but not in adults. The rapid development of normoglycemia in pups with insulin minipumps, compared with pups injected daily with the same dose, suggests that continuous early insulin may produce insulin resistance.     

Functional rice centromeres are marked by a satellite repeat and a centromere-specific retrotransposon

The centromere of eukaryotic chromosomes is essential for the faithful segregation and inheritance of genetic information. In the majority of eukaryotic species, centromeres are associated with highly repetitive DNA, and as a consequence, the boundary for a functional centromere is difficult to define. In this study, we demonstrate that the centers of rice centromeres are occupied by a 155-bp satellite repeat, CentO, and a centromere-specific retrotransposon, CRR. The CentO satellite is located within the chromosomal regions to which the spindle fibers attach. CentO is quantitatively variable among the 12 rice centromeres, ranging from 65 kb to 2 Mb, and is interrupted irregularly by CRR elements. The break points of 14 rice centromere misdivision events were mapped to the middle of the CentO arrays, suggesting that the CentO satellite is located within the functional domain of rice centromeres. Our results demonstrate that the CentO satellite may be a key DNA element for rice centromere function.     

Dietary input of microbes and host genetic variation shape among-population differences in stickleback gut microbiota

To explain differences in gut microbial communities we must determine how processes regulating microbial community assembly (colonization, persistence) differ among hosts and affect microbiota composition. We surveyed the gut microbiota of threespine stickleback (Gasterosteus aculeatus) from 10 geographically clustered populations and sequenced environmental samples to track potential colonizing microbes and quantify the effects of host environment and genotype. Gut microbiota composition and diversity varied among populations. These among-population differences were associated with multiple covarying ecological variables: habitat type (lake, stream, estuary), lake geomorphology and food- (but not water-) associated microbiota. Fish genotype also covaried with gut microbiota composition; more genetically divergent populations exhibited more divergent gut microbiota. Our results suggest that population level differences in stickleback gut microbiota may depend more on internal sorting processes (host genotype) than on colonization processes (transient environmental effects).